PROJECT SUMMARY/ABSTRACT The long-term objectives are to understand the mechanism of mechanotransduction in auditory hair cells and delineate the factors underlying the cochlea's tonotopic organization. Experiments will focus on the attributes and molecular composition of the mechanotransducer (MET) channel and its modulation by Ca2+, with the goal of collecting and assessing evidence that transmembrane channel-like protein isoforms TMC1 and TMC2 form a central component of the channel that dictates its biophysical properties. Hair cell responses will be measured in acutely isolated cochleas of wild-type and mutant mice. Organotypic cochlear cultures will also be used, some of which will be modified by viral transfection with mutated components of the transduction machinery. Specific aims are: (1) to record and analyze the substructure of single MET channel currents in hair cells at different cochlear locations, and combine the results with imaging of endogenous fluorophore-tagged transmembrane channel-like proteins, TMC1 and TMC2. The hypothesis is that the tonotopic increase in channel conductance from apex to base is determined by increasing numbers of TMC1-associated channels gated cooperatively; (2) to measure MET channel adaptation in Tmc1 and Tmc2 mutants, comparing the speed and Ca2+ dependence, and testing the hypothesis that adaptation is controlled by intracellular Ca2+, which will be manipulated by photolysis of caged Ca2+. Adaptation will also be characterized in mice with engineered Tmc1 mutations; (3) to determine the function of CIB2 (calcium and integrin binding protein 2), which is known to associate with TMC1 and is mutated in certain genetic deafnesses. We will use short peptides to interfere with the interaction with TMC1, testing the hypothesis that CIB2 is involved in MET channel adaptation; (4) to develop approaches to expressing TMC1 in non-hair cells, and testing for its performance as a mechanically gated ion channel. This will include investigating cell lines that endogenously express TMC1; and transfecting these cell lines with TMC1 possessing tags to follow its interaction with microtubules and trafficking to the plasma membrane; methods for isolating new chaperones that facilitate trafficking of TMC1 to the plasma membrane, will be developed; (5) to define the function of the hair-cell Ca2+- binding protein sorcin, which is known to regulate ryanodine receptors in cardiac muscle; the hypothesis is that modulation of calcium-induced calcium release by ryanodine receptors is fundamental to the efferent cholinergic synapse, and also to outer hair cell electromotility, via sub-membranous cisternae. It is hoped that the results will supply evidence on the molecular composition of the hair cell transduction apparatus, and will yield information about proteins that are mutated in certain forms of human genetic deafness.